JP6718450B2 - Chimeric antigen receptor and method of using the same - Google Patents

Description

Most T cells express the αβ T cell receptors (TCRs) and recognize antigens in the form of peptides (epitopes) presented by the major histocompatibility complex (MHC) on other cells. The TCRs of cytotoxic T lymphocytes recognize epitopes presented by MHC class I molecules on the surface of almost every cell in the body. The TCRs of helper T cells recognize epitopes presented by MHC class II molecules on the surface of antigen presenting immune cells including macrophages, dendritic cells and B cells. Efficient recognition of epitopes by T cells also involves T cell surface glycoproteins: CD8 on cytotoxic T lymphocytes (CD8 ⁺ T cells) that bind to MHC class I and class II molecules, respectively. , And CD4 on helper T cells (CD4 ⁺ T cells). Binding of TCR to an epitope can signal the nucleus of T lymphocytes and induce a T cell response.

The clear and efficient identification of T cell antigen specificity holds great potential for the development of efficient immunotherapy and diagnostic tools. Especially:
-May be useful in assessing the safety of the T cell therapy employed;
-It may enable new approaches in the treatment of autoimmune diseases, cancer and in the development of new vaccines.

However, TCRs bind the MHC-peptide complex with low affinity, which renders the methods of phage and yeast display inefficient. Alternative methods, such as position-scanning combinatorial peptide libraries, take advantage of the cross-reactivity of TCRs and use peptide pools to define motifs that result in T cell activation. Apart from similar affinity constraints, these cumbersome methods suffer a high probability of false positive results. Due to the examination of random peptide sequences, the identified peptide motifs are not clear or have no clear homology with the native protein.

The problem underlying the present invention is to provide a method for the direct and sensitive, unbiased identification of antigenic peptide specificity of CD4 ⁺ and CD8 ⁺ T cells for use in vitro and in vivo. Is. This problem is solved by the subject matter of the independent claims.

FIG. 1 shows the structure and antigen-specific reactivity of the MCR2 sensor (chimeric antigen receptor polypeptide heterodimer with extracellular portion derived from MHC II). FIG. 1a shows a schematic representation of MCR2 and the interaction of MCR2 with a peptide-specific TCR. FIG. 1b shows the expression of MCR2 (gp61) on the surface of BEKO thymocytes. FIG. 1c is down-regulation of MCR2 (gp61) (left panel) or MCR2 (OVA) (right panel) in BEKO cells co-cultured with Gp61-specific Smarta or OVA-specific OT-II TCR transgenic CD4 ⁺ T cells. Shows the time course of. Values represent MCR2 levels as a percentage of mean fluorescence intensity at the start of co-culture. FIG. 1d shows peptide-specific NFAT activity (GFP expression) in H18.3.13 cells transduced with MCR2 (gp61) or MCR2 (OVA) co-cultured with Smarta or OVA-specific OT-II CD4 ⁺ T cells. Shows the time course of. The histogram shows an example of NFAT activation measurements in MCR2(gp61) ⁺ cells at the indicated time points. FIG. 1e shows the sensitivity of peptide-specific reporter cells. NFAT after dilution of MCR2(gp61) ⁺ H18.3.13 cells with MCR2(OVA) ⁺ H18.3.13 cells and co-culture with untreated (triangles) or Smarta CD4 ⁺ T cells (squares). The activity was measured. The graph shows a linear correlation (R>99) between the percentage of GFP ⁺ H18.3.13 cells and the percentage of cells with MCR2 (gp61). FIG. 1f shows the minimal frequency of peptide-specific T cells capable of inducing robust NFAT activation in MCR2 ⁺ reporter cells. Splenocytes from the Smarta and OT-II transgenic mice were mixed at different ratios and used to stimulate MCR2(gp61) ⁺ or MCR2(OVA) ⁺ H18.3.13 cells. Graph shows GFP ⁺ cells in MCR2(gp61) ⁺ H18.3.13 or MCR2(OVA) ⁺ H18.3.13 cells as a function of percentage of peptide-specific CD4 ⁺ T cells after 8 hours of co-culture. Shows the ratio of. FIG. 1g shows the minimum number of peptide-specific T cells able to elicit a response in reporter cells. NFAT activation in MCR2(gp61) ⁺ (left panel) and MCR2(OVA) ⁺ (right panel) H18.3.13 cells co-cultured with different numbers of Smarta or OT-II CD4 ⁺ cells overnight. FIG. 1h shows the kinetics of proliferation of LCMV-specific CD4 ⁺ T cells in the blood of infected mice (n=3) detected by activation of MCR2 (gp61) ⁺ H18.3.13 cells. The percentage of GFP ⁺ reporter cells after overnight co-culture with blood taken on different days post infection is shown. Blood from untreated Smarta mice was used as a positive control. FIG. 2 shows the screening of the gp61 mimotope. FIG. 2a shows a scheme for RAG-mediated peptide randomization based on the construction of an RSS recombination signal sequence, MCR2(gp61-RSS) mimotope library (see Materials and Methods). Figure 2b is an example of a modified gp61 sequence found in the library (new amino acid sequence is shown in grey). FIG. 2c: Transduction of 16.2c11 cells with NFAT-FT reporter with MCR2 (gp61-RSS) library or MCR2 (gp61) or MCR2 (OVA) as controls, and Smarta or OT-II CD4 ⁺ T. Co-culture with cell hybridomas. Single MCR2(gp61-RSS) ⁺ cells showing NFAT activation (blue FT fluorescence) after 9 hours co-culture with Gp61-specific Smarta hybridoma (upper right panel) were isolated and expanded. FIG. 2d shows activation of MCR2(gp61) ⁺ 16.2c11 cells, MCR2(gp61S) ⁺ 16.2c11 cells and MCR2(gp61N) ⁺ 16.2c11 cells co-cultured with a Smarta hybridoma (upper right panel). Figure 2e shows the sequences of two new gp61 mimotopes found in the original gp61 and MCR2 (gp61-RSS) libraries. FIG. 2f shows CFSE (histogram) diluted with Smarta CD4 ⁺ T cells after 3 days of co-culture with dendritic cells pulsed with different concentrations of gp61 mimotope and cytokine production (dot plot) at a concentration of 100 nM. Figure 3 shows the search for new LCMV epitopes. FIG. 3a shows CD4 ⁺ T cells from LCMV-infected mice as p. i. Purified from day 5 or day 8 spleens and fused with BW5147 cells carrying the NFAT-GFP reporter. The reactivity of the resulting hybridomas was determined by the expression of GFP after co-culture with dendritic cells pulsed with LCMV or gp61. FIG. 3a is a histogram of an example of different types of reactivity (LCMV-no color or gp61-dark gray color in). FIG. 3b shows CD4 ⁺ T cells from LCMV-infected mice as p. i. Purified from day 5 or day 8 spleens and fused with BW5147 cells carrying the NFAT-GFP reporter. The reactivity of the resulting hybridomas was determined by the expression of GFP after co-culture with dendritic cells pulsed with LCMV or gp61. Figure 3b is a table summarizing the data. Figure 3c shows a scheme of a cloning strategy for constructing a library enriched for naturally occurring peptides (NPLs). The ORF-open reading frame directs the sequence of the native protein. FIG. 3d shows co-culture of MCR2(LCMV-NPL) ⁺ 16.2c11 reporter cells with LCMV-reactive hybridomas of unknown peptide specificity (H2, H3, H14 and H30). Activated reporter cells were isolated, expanded and co-cultured again with the corresponding hybridomas. This operation was repeated 3 times in order to achieve sufficient concentration. The dot plot shows an example of NFAT activation after 2-3 co-cultures. Figure 3e, after the third enrichment, single cells were isolated, expanded and their reactivity was confirmed by co-culture with hybridomas (histogram shows an example of NFAT reactivity). The table on the right shows the frequency of reactive clones. Peptide sequences from the MCR2 construct representing the major NP311 epitope and the new NP547 epitope are shown below. FIG. 4 shows a functional test of MCR1 (MHCI; chimeric antigen receptor polypeptide heterodimer with extracellular portion from gp33/H2-Hb) molecules transduced into the H18.3.1 reporter cell line. .. (A) After overnight culture in wells coated with αMHC-I antibody, NFAT activation was induced in MCR-1 transduced cells but not in control cells. FIG. 5a shows activation of MCR2(LCMV-NPL) ⁺ 16.2c11 cells after co-culture with hybridomas H3 and H4 once, twice, and three times. FIG. 5b shows an example of activation of the MCR2 ⁺ 16.2c11 clone from the screening of a random peptide MCR2 library with hybridoma H9 after the final co-culture. An example of sequences recovered from the library before and after concentration of H9-reactive peptide is shown. FIG. 6 shows the peptide-specific reactivity and sensitivity of the MCR2 sensor. FIG. 6a shows peptide-specific NFAT activation (GFP reporter expression) in MCR2(OVA) ⁺ H18.3.13 cells co-cultured with Smarta CD4 ⁺ T cells as a control or with OT-II CD4 ⁺ T cells. Indicates a time course. Figure 6b shows the sensitivity of peptide-specific reporter cells. MCR2(OVA) ⁺ H18.3.13 cells were diluted with MCR2(gp61) ⁺ H18.3.13 cells and NFAT activation was measured after co-culture with OT-II CD4 ⁺ T cells. The graph shows a linear correlation (R>0.99) between the percentage of GFP ⁺ H18.3.13 cells and the percentage of cells with MCR2.

Terms and Definitions Amino acid sequences are given from the amino terminus to the carboxylic acid terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd edition p.21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

In the context of the present invention, the term identity or sequence identity is used in its sense known in the field of genetics and bioinformatics; they are single quantitative representations of position-by-position sequence comparison results. Refers to a parameter. Methods of sequence comparison are known in the art; the published BLAST algorithm is an example.

For amino acid sequence comparisons, one such example is the BLASTP algorithm using default settings such as: Expected threshold: 10; Word size: 3; Maximum match within query range: 0; Matrix: BLOSUM62; Gap cost: Existence 11, Expansion 1; Configuration adjustment: Conditional configuration score matrix adjustment. In the absence of further details, these settings are used for the determination of amino acid identity values shown below.

For comparison of nucleic acid sequences, one such example is the BLASTN algorithm using default settings such as: Expected threshold: 10; word size; 28; maximum match within query: 0; match / Mismatch score: 1. -2; Gap cost: linear. In the absence of further details, these settings are used in the determination of nucleic acid sequence identity values shown below.

In the context of the present specification, the term major histocompatibility complex (MHC) is used in its sense known in the field of cell biology and biochemistry; it is a suitable method for recognition by the T cell receptor. And refers to a cell surface molecule that presents a peptide that is a fragment of a protein. Peptides recognized by the immune system are referred to in the present context as epitopes or antigenic peptides or oligopeptides.

There are two major classes of MHC molecules: class I and class II.

MHC class I exists as an α chain containing three sites of α1, α2, and α3. The α3 site interacts with the non-MHC molecule β2 microglobulin. The displayed or presented peptides are retained by the peptide binding groove in the central part of the α1/α2 heterodimer. The α3 subunit contains a transmembrane site that anchors the MHC class I molecule to the cell membrane.

MHC class II is formed of two chains, α and β, and has two sites, α1 and α2 and β1 and β2, respectively. The peptide-binding groove, the structural element formed by MHC class II molecules that presents or displays peptide epitopes, is formed by α1 and β1 heterodimers. The α2 and β2 subunits contain a transmembrane site that anchors MHC class II molecules to cell membranes.

In their immature form, MHC class I and class II molecules contain a signal peptide, such as the majority of new synthetic proteins, which directs them to the secretory pathway. The MHC signal peptide sequence is located upstream (5') of the MHCα1 site on the MHC mRNA molecule. After cleaving the signal peptide, the MHC molecule is called the mature MHC molecule.

In the context of the present specification, the term β2 microglobulin site is used in its sense known in the field of cell biology and biochemistry; it is a non-MHC which is part of an MHC class I heterodimer molecule. Refers to a molecule. In other words, it constitutes the β chain of MHC class I heterodimers.

In the context of the present specification, the term T cell receptor (TCR) is used in its sense known in the field of cell biology and biochemistry; it refers to an antigen that binds to a major histocompatibility complex molecule. Refers to a molecule found on the surface of T cells that can be recognized. TCRs are disulfide-bridged membrane-anchored heterodimers composed of highly variable α and β chains. Each chain contains two extracellular positions, a variable (V) region and a constant (C) region. The variable region binds to the peptide-MHC complex; the constant region is proximal to the cell membrane, followed by a hinge region, a transmembrane region and a short cytoplasmic tail.

In the context of the present specification, the term T cell receptor complex is used in its sense known in the field of cell biology and biochemistry; the term refers to two heterologous CD3ε/δ and CD3γ/ε. It refers to the dimeric signal molecule, as well as the octameric complex of the CD247ζ/ζ (known as the TCRζ chain or zeta chain) homodimer and the heterodimer TCRα/β. The ionizable residues in the transmembrane site of each subunit form a polar network of interactions that holds the complex together. The cytoplasmic tail of TCRα/β is so short that it is not involved in signal transduction and these signaling molecules are essential in the signal transduction of induced TCRs into cells. The most common mechanism for molecular activation or regulation downstream of the cytoplasmic membrane is through phosphorylation/dephosphorylation by protein kinases. The intracellular portion of CD3 and CD247ζ contains an immunoreceptor tyrosine-based activation motif (ITAM) targeted by the Src family of tyrosine kinases.

In the context of the present specification, the term operably linked refers to the association of the active states of two different functions. For example, the receptor polypeptide (function 1) may be operably linked to a reporter gene and its promoter (function 2); and, if the receptor changes (eg, activates) its activation state, The promoter of the reporter gene also changes its activation state (eg activation) and transcribes the reporter gene. One such non-limiting example is the activated T cell nuclear factor (NFAT) signaling pathway known in the art. Activation of native TCRs in T cells (function 1) results in activation of protein kinases and phosphatases that initiate nuclear translocation of NFAT transcription factors leading to expression of NFAT target genes (function 2). In other words, the TCR is operably linked to the expression of the NFAT target gene.

In the context of the present specification, the term activation of a reporter gene refers to a change in the active state of the reporter gene. One example of such a change in the activation state is activation of the promoter of the reporter gene. The activation results in increased transcription/translation of the reporter gene. In another example, cleavage of the reporter protein inhibitor results in an increase in the amount of active reporter protein.

In the context of the present specification, the term transgenic is used in its sense known in the field of cell biology; the term is endogenously possessed by an exogenous nucleic acid sequence introduced into it. Not referring to exhibiting new characteristics.

In the context of the present specification, the term antigen receptor is used in its sense known in the field of cell biology and immunology; the term refers to a surface receptor capable of binding an antigen or an epitope. Examples of antigen receptors are B cell receptors and T cell receptors.

In the context of the present specification, the term chimeric antigen receptor refers to an artificially designed receptor that has a portion of the antigen receptor. A non-limiting example is a hybrid receptor comprising a T cell receptor or a B cell receptor-derived site fused to an MHC site and/or an antigenic peptide sequence.

In the context of the present specification, the term oligopeptide or oligopeptide sequence refers exclusively to a T cell active oligopeptide, which when presented with respect to an MHC molecule on a cell is a cognate T cell receptor. Can be elicited by a T cell response. When referring to the oligopeptide sequences comprised in the (longer) polypeptides of the invention, those skilled in the art will understand that they refer to oligopeptide sequences that can be recognized by the TCR when presented on an MHC molecule. Ah In addition to the MHC presenting T cell active peptide, the oligopeptide sequence also contains various amino acid linkers that allow the THC active peptide to be adapted to the MHC molecule. Examples of linker lengths and sequences are shown below.

In a first aspect of the invention there is provided a method for the identification of a peptide sequence recognized by a TCR, the method comprising the steps of:
i. Is to provide multiple mammalian cells, where
Each of the plurality of mammalian cells expressing a member of the library,
Each member of the library encodes a transgenic antigen receptor molecule,
-The transgenic antigen receptor molecule is
-The extracellular position of MHC1 or MHC2,
• In the polypeptide sequence of the extracellular sequence (the term in sequence means that the oligopeptide may be included either between the N and C boundaries of the extracellular sequence or at each of its ends). ,) wherein the oligopeptide is different for each member of the library, and wherein the oligopeptide is presented in a manner suitable for recognition by the T cell receptor,
・Transmembrane site of T cell receptor (TCR), and ・Intracellular site of TCR,
Including
-The transgenic antigen receptor molecule is operably linked to a reporter protein, whereby binding of an allogeneic T cell receptor to the transgenic antigen receptor results in activation of the reporter gene.
ii. Contacting a plurality of mammalian cells with a preparation of T lymphocytes capable of binding via a T cell receptor expressed on the surface of T lymphocytes to an oligopeptide sequence contained in the transgenic antigen receptor.
iii. Separating cells having a detectable reporter protein from the plurality of mammalian cells according to the level of the detectable reporter protein to obtain activated cells.
iv. Isolating DNA encoding a library member expressed from the activated cells.
v. Sequencing the oligopeptide sequences contained in the transgenic antigen receptor expressed in the activated cells. The determined sequence of the oligopeptide is the recognizable peptide sequence of the TCR.

In other words, the method of the invention may assess the potential of an oligopeptide as an antigen, or, more precisely, the potential of a particular oligopeptide sequence to become an MHC-presenting epitope that elicits an allogeneic T cell response. Allows you to evaluate. To achieve this evaluation, a library of transgenic antigen receptors containing potentially antigenic oligopeptides is required. The members of the library are expressed in each mammalian cell of the plurality of cells, where the oligopeptide sequences can be different for each member of the library. The oligopeptide is presented by the transgenic antigen receptor in a manner suitable for recognition by the T cell receptor. Binding of antigen oligopeptides by the provided T lymphocytes through their T cell receptor activates a reporter protein that is operably linked to the transgenic antigen receptor. The mammalian cells are separated according to the level of detectable reporter. DNA is isolated from the separated mammalian cells and the oligopeptide DNA contained is sequenced. All oligopeptides recovered by this method are recognized by the T cell receptor.

The transgenic antigen receptor is an oligopeptide presented by a method suitable for recognition by a T cell receptor, an extracellular site of MHC1 or MHC2, a transmembrane site of the T cell receptor and an intracellular site of the T cell receptor. Including. The oligopeptide and the different site are covalently linked to form a single polypeptide chain (extracellular-transmembrane-intracellular). In one embodiment, the transgenic condition further comprises the hinge region of the T cell receptor located between the extracellular position and the transmembrane site.

In the context of the present specification, the term expressing or expression is used in its sense in the field of cell biology and molecular biology; the term refers to the transcription and translation of DNA sequences and the mRNA from which they are derived.

In one embodiment, step iii. Separation of cells having a detectable reporter protein from a plurality of mammalian cells according to the level of detectable reporter protein according to claim 1, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or It is carried out 10 times. This embodiment is particularly advantageous when using high complexity libraries.

According to another aspect of the invention, there is provided a method for the identification of oligopeptide sequences recognizable by a TCR. The method includes:
i. To provide mammalian cells, where
-The mammalian cell expresses a transgenic antigen receptor molecule,
-The transgenic antigen receptor molecule is
An extracellular position of MHC1 or MHC2 containing an oligopeptide presented in a manner suitable for recognition by the T cell receptor,
-Including the transmembrane site of the T cell receptor (TCR), and-the intracellular site of the TCR,
-The transgenic antigen receptor molecule is operably linked to a reporter protein, whereby binding of the T cell receptor to the transgenic antigen receptor results in activation of said reporter gene.
ii. Contacting a mammalian cell with a preparation of T lymphocytes capable of binding to an oligopeptide contained in a transgenic antigen receptor via a T cell receptor expressed on the surface of T lymphocytes. Binding of the T cell receptor to the transgenic antigen receptor activates the reporter gene, resulting in activated mammalian cells.
iii. DNA encoding a library member expressed from the activated mammalian cell is isolated.
iv. The oligopeptide sequences contained in the transgenic antigen receptor are sequenced.

In one embodiment, the transgenic antigen receptor further comprises a hinge region.

In one embodiment according to the first and second aspects of the invention, the transgenic antigen receptor is a transgenic antigen receptor already known in the art. Examples of suitable transgenic antigen receptors known in the art are Jyothi et al., Nat Biotechnol 20, 1215-1220 (2002); Geiger et al., Blood 98(8) 2364-2371 (2001); US20082866312A1; Mekala et al. PNAS 102(33), 11817-11822, (2005);. Moisini et al., J Immunol (2008), 180:3601-3611; Scott et al., J Automm 35, 390-397 (2010).

In one embodiment of any of the above aspects of the invention the transgenic antigen receptor is a chimeric antigen receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, and a. The first polypeptide comprises an extracellular position of the major histocompatibility complex I (MHC class I) α chain, and the second polypeptide comprises a β2 microglobulin site, or b. The first polypeptide comprises the extracellular position of the major histocompatibility complex II (MHC class II) α chain, and the second polypeptide is the major histocompatibility complex II (MHC class II). Contains the extracellular portion of the β chain.

At least one of the first and second polypeptide chains further comprises an oligopeptide covalently linked to an extracellular MHC molecule site, wherein the oligopeptide is recognized by the T cell receptor.

In one embodiment the oligopeptide sequence further comprises a linker sequence of 4 to 16 amino acids, in particular 6, 8, 10, 12 or 14 amino acids in length. In one embodiment, the linker is mainly composed of glycine and one of serine, threonine and alanine. In one embodiment, the linker comprises glycine and serine only.

One of the first polypeptide and the second polypeptide further comprises an intracellular site or intracellular tail of the T cell receptor α chain, a transmembrane site and a hinge region, and the first polypeptide and the second polypeptide The other of the two polypeptides contains the intracellular portion of the T cell receptor β chain, the transmembrane region and the hinge region.

In one embodiment, the oligopeptide sequence and the glycine-serine linker are inserted between the last amino acid of the MHC signal/leader peptide and the first amino acid of the MHC α1 or β1 site.

In one embodiment, the oligopeptide sequence and the linker sequence are inserted 1, 2, 3, 4 or 5 amino acids after the MHC α1 site sequence or β1 site sequence. Binding of the peptide to the β chain (β1 site) will insert the peptide into the MHC molecule in the orientation most commonly found. The peptide is inserted into the α chain in the opposite direction.

In one embodiment, the extracellular portion of the MHC molecule is available to those skilled in the art in specialized databases such as IMGT/HLA (http://www.ebi.ac.uk/ipd/imgt/hla/), It is selected from the human major histocompatibility complex family HLA. Alternatively, the HLA sequence is derived from RNA extracted from a patient blood or tissue sample.

In one embodiment, the oligopeptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acids (AA) in length. .. The majority of MHC class I peptides are 8-10 amino acids in length, while MHC class II is able to present longer peptides due to the open binding gap, yet the actual epitope is It is in the range of 10 to 12 AA. In the method disclosed herein, the peptide may be longer than 20 (see Figure 3, LCMV peptides found in the method of the invention include 24AA). In one embodiment, larger oligopeptides are utilized, which allows for the screening of more binding registers on one peptide.

In one embodiment, the intracellular portion, the transmembrane portion and the hinge region of the transgenic antigen receptor in the first and/or second polypeptide are not derived from the same TCR chain. In other words, the hinge region or transmembrane site of the TCR α chain can be linked to the transmembrane site or intracellular site of the TCR β chain and vice versa.

In one embodiment the chimeric antigen receptor polypeptide heterodimer according to the second aspect of the invention is at least 80%, 85%, 90%, 92%, 94%, 95% relative to SEQ ID NO:007. %, 96%, 97%, 98% or at least 80%, 85%, 90%, 92%, 94% with respect to the first polypeptide having an amino acid sequence having an identity of 99% and SEQ ID NO:008 , A second polypeptide having an amino acid sequence having 95%, 96%, 97%, 98% or 99% or greater identity.

According to a third aspect of the present invention there is provided a chimeric antigen receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide. The first polypeptide is linked to the second polypeptide by one or more disulfide bonds, and a. The first polypeptide comprises the extracellular portion of the major histocompatibility complex I (MHC class I) α chain, and the second polypeptide is the major histocompatibility complex I (MHC class I). Contains a relevant β2 microglobulin site, or b. The first polypeptide comprises the extracellular position of the major histocompatibility complex II (MHC class II) α chain, and the second polypeptide is the major histocompatibility complex II (MHC class II) β. Including chains.

One of the first polypeptide and the second polypeptide further comprises a hinge region of a T cell receptor (TCR) α chain, a transmembrane site (and an intracellular site or tail), and the first polypeptide The other of the peptide and the second polypeptide comprises the hinge region of the T cell receptor (TCR) β chain, the transmembrane site (and intracellular site or tail).

In other words, the chimeric antigen receptor polypeptide heterodimer is capable of presenting an oligopeptide (epitope) in the MHC environment such that the polypeptide (epitope) is recognized and bound by its cognate TCR. Binding of the cognate TCR results in activation of the intracellular sites of CD3 and CD247 associated with the chimeric antigen receptor polypeptide heterodimer, which results in activation of NFAT.

In one embodiment, the extracellular position of the MHC molecule is
i. MHC class I α1 and α2 and α3 sites of the first polypeptide (in addition to β2 microglobulin of the second polypeptide), or ii. MHC class II α1 and α2 sites of the first polypeptide, and MHC class II β1 and β2 sites of the second polypeptide,
including.

In one embodiment, the first polypeptide comprises substantially the entire extracellular position of the major histocompatibility complex I (MHC class I) α chain. In one embodiment, the first polypeptide is an extracellular position of major histocompatibility complex I (MHC class I).

In one embodiment, the second polypeptide comprises substantially the entire extracellular position of major histocompatibility complex I (MHC class I) associated with β2 microglobulin. In one embodiment, the second polypeptide is major histocompatibility complex I (MHC class I) associated with β2 microglobulin.

In another embodiment, the first polypeptide comprises substantially the entire extracellular position of the major histocompatibility complex II (MHC class II) α chain. In one embodiment, the first polypeptide is the extracellular position of the major histocompatibility complex II (MHC class II) α chain. In one embodiment, the second polypeptide comprises substantially the entire major histocompatibility complex II (MHC class II) β chain. In one embodiment, the second polypeptide is the major histocompatibility complex II (MHC class II) β chain.

In one embodiment, at least one of said first and second polypeptide chains further comprises an antigenic peptide covalently linked to an MHC extracellular site, wherein said oligopeptide is recognized by a T cell receptor. be able to.

In one embodiment, the antigenic peptide sequence and the glycine-serine linker are inserted between the last amino acid of the MHC signal/leader peptide and the first amino acid of the MHC α1 or β1 site.

In one embodiment, the antigen-peptide sequence and the linker sequence are inserted 1, 2, 3, 4 or 5 amino acids after the MHC α1 site sequence or β1 site sequence.

In one embodiment, the extracellular portion of the MHC molecule is human major histocompatibility available in specialized databases such as IMGT/HLA (http://www.ebi.ac.uk/ipd/imgt/hla/). It is selected from the gene complex family HLA. Alternatively, the HLA sequence is derived from RNA extracted from a patient blood or tissue sample.

In one embodiment, the oligopeptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acids in length. The majority of MHC class I peptides are 8-10 amino acids long, while MHC class II has the ability to present longer peptides due to the open gaps in binding, yet the actual epitope. Is within 10 to 12 AA. In the method disclosed herein, the peptide may be longer than 20 (see Figure 3 LCMV peptides found in the method of the invention include 24AA). Having a larger oligopeptide is advantageous in that it can screen more binding registers on a single peptide.

In one embodiment, the hinge region, transmembrane site and extracellular portion of the chimeric antigen receptor polypeptide in the first and/or second peptide are not derived from the same TCR chain. In other words, the hinge region of the TCR α chain or the transmembrane site can be linked to the transmembrane site or intracellular site of the TCR β chain, and vice versa.

According to a fourth aspect of the present invention, a nucleic acid molecule encoding the chimeric antigen receptor polypeptide heterodimer according to the third aspect of the present invention, particularly a nucleic acid having a promoter sequence that can be carried out in a mammalian host cell. A molecule is provided.

According to a fifth aspect of the present invention there is provided a cell, in particular a mammalian cell comprising or expressing the nucleic acid molecule according to the fourth aspect of the present invention.

According to a sixth aspect of the invention there is provided a cell, in particular a mammalian cell, more particularly a mammalian T lymphocyte,
i. The chimeric antigen receptor polypeptide according to the fifth aspect of the invention ii. An effector function operably linked to the chimeric antigen receptor polypeptide heterodimer,
including.

In one embodiment, the effector function is as follows:
i. A reporter protein, and/or ii. Ability to induce cell death, particularly apoptosis, in cells bound to the chimeric antigen receptor polypeptide heterodimer.

In one embodiment, the reporter protein is selected from:
i. Fluorescent protein,
ii. Luciferase protein,
iii. Antigen resistance gene,
iv. Cre recombinase,
v. CAS-9 nuclease, or vi. CAS-9 chimeric transcription repressor or transcriptional activator.

According to a seventh aspect of the present invention there is provided a method for obtaining a preparation of T lymphocytes having a reduced reactivity with an antigen. The method includes:
i. Providing a preparation of T lymphocytes obtained from a patient,
ii. Contacting the mammalian cell according to the fifth or sixth aspect of the invention with a T lymphocyte preparation,
Here, the mammalian cell is characterized in that the effector function is capable of inducing cell death, particularly apoptosis, in a cell bound to the chimeric antigen receptor polypeptide heterodimer.

In other words, specific T lymphocytes capable of binding the oligopeptide sequences provided within the chimeric antigen receptor polypeptide heterodimer of mammalian cells are conferred cell death, in particular apoptosis.

According to an eighth aspect of the invention, there is provided a method for reducing T lymphocytes active against a particular antigen for use in a patient in need thereof. The method comprises the following steps:
i. Providing a preparation of mammalian cells according to the fourth aspect of the invention,
ii. Administering the mammalian cells to a patient,
Here, the mammalian cell is characterized in that the effector function is capable of inducing cell death, in particular apoptosis, of a cell bound to the chimeric antigen receptor polypeptide heterodimer.

In other words, it selectively reduces T lymphocytes capable of binding to the oligopeptide sequences presented in the chimeric antigen receptor polypeptide heterodimer. It can be used, for example, for the reduction or elimination of autoreactive T lymphocytes in patients in need thereof.

In one embodiment, this method is used to treat an autoimmune disease such as allergy. In one embodiment, this method is used for the prevention and treatment of organ rejection after transplantation.

According to a ninth aspect of the invention, the mammalian cells according to the fourth aspect of the invention include, but are not limited to, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Crohn's disease and inflammatory bowel. Provided for use in the treatment or prevention of immune dysfunction, including syndromes, transplant rejection, or autoimmune disease. In other words, the mammalian cells are used for the reduction of T lymphocytes that have activity against antigens associated with particular diseases.

According to a tenth aspect of the invention, there is provided a method for detecting an immune response of a patient to an oligopeptide. The method comprises the following steps:
i. A mammalian cell according to the fourth aspect of the invention is provided, wherein the cell comprises a reporter protein operably linked to the chimeric antigen receptor polypeptide heterodimer,
ii. Providing an ex vivo patient blood sample containing T lymphocytes associated with an immune response,
iii. Contacting a mammalian cell with a blood sample under conditions that enable effector function, and
iv. Detects reporter protein in mammalian cells.

In other words, if the T lymphocytes capable of binding to the oligopeptide used were enriched in the blood of the individual, resulting in the expression of a strong reporter protein, this would result in an immune response to the oligopeptide. Represent

In one embodiment, the oligopeptide used in this method is from a virus, bacterium, fungus or parasite.

In one embodiment, the method according to the ninth aspect of the invention comprises
i. Separating mammalian cells according to expression of the reporter protein,
ii. Isolating DNA from the separated mammalian cells, and iii. Determining the sequence of the oligopeptide encoded by the chimeric antigen-receptor polypeptide heterodimer according to the fifth aspect of the invention,
The process of is further included.

In other words, a plurality of mammalian cells according to the fourth aspect of the invention having different oligopeptides in the chimeric antigen-receptor polypeptide heterodimer are used. Each of these oligopeptides is derived from a particular pathogen, allergen, tumor or inflamed tissue (in the case of autoimmune patients). This allows for simultaneous testing for many different immune responses. One non-limiting example is its use as a diagnostic tool.

For example, where alternatives for a single separable feature, such as linker sequence, linker length, are referred to herein as "embodiments", such alternatives are contemplated by the inventions disclosed herein. May be freely combined from the individual embodiments of. Those skilled in the art will appreciate that the isolated features of the invention described as particular embodiments may be combined with any other feature described.

The invention can be further explained by the following examples and figures, whereby further embodiments and advantages are described. These examples illustrate the invention but do not limit its scope.

Example 1: Screening T cell epitopes in mammalian cells using the disclosed chimeric antigen receptor polypeptide heterodimers

Current methods for identifying allogeneic T cell epitopes are in principle based on two main approaches. The first approach is to stain physical MHC-by staining T cells with MHC tetramers or by staining phage, yeast or insect cells displaying peptide-MHC complexes with recombinant TCRs. It relies on detecting TCR interactions. The second approach relies on the measurement of T cell activation in peptide pools or in co-culture with dendritic cells (DCs) displaying position-scanning combinatorial peptide libraries. Although screening MHC tetramer libraries is effective in determining the fine specificity of known or predicted antigen recognition, not all peptide-MHC tetramers bind with equal force. because it is not a low-affinity interactions are overlooked easily (e.g., for dyeing similarly OVA-specific OT-II and gp61 specific Smarta2 T cells individually, ^{OVA-I-a b} tetramer or more is required 400 times the ^{gp61-I-a b} tetramer). Similar affinity constraints apply to current peptide-MHC display methods and soluble TCRs are used. Furthermore, the MHC molecule needs to be mutated to allow efficient surface expression on phage or yeast cells. Screening of position-scanning combinatorial peptide libraries takes advantage of the cross-reactivity of TCRs and uses peptide pools to determine motifs that result in T cell activation. Although T cell epitopes similar to naturally occurring peptides have been found in this way, the identified peptides often do not have clear homology with known proteins and require the assistance of bioinformatics approaches ..

We disclose herein the development of a universal system that allows for direct, unbiased, sensitive and efficient epitope screening in mammalian cells. Such methods: i) provide a complex mixture of APCs, each presenting a peptide of a unique naturally occurring sequence; ii) an efficient method for identifying and isolating APCs presenting a cognate peptide. Iii) provides the possibility of iteratively repeating the procedure, and iv) allows the peptide sequence to be readily recovered by cloning. In order to produce APCs that meet the first criterion, the inventor followed the approach of creating "single peptide" mice and constructed different peptide-MHC presentation systems. Recombinant DNA technology allowed the peptide to bind directly to the MHC molecule, creating a stable complex and preventing other peptides from binding. Transfection of such a peptide-MHC complex library into MHC-deficient cells results in a pool of cells displaying unique peptides (see Materials and Methods for details). Ideally, identification of APCs bearing cognate peptides for a particular T cell would require a signal that would be readily measurable once the peptide-MHC complex binds to a specific T cell TCRs. And Thus, the peptide-MHC fusion molecule binds to a TCR complex that has been adjusted to sense low affinity interactions. Direct zeta chain (CD247) fusions have been successfully used to construct a variety of chimeric antigen receptors. However, in order to create a molecular sensor that mimics the natural TCR complex as closely as possible, the peptide MHC complex uses truncated TCRα and TCRβ consisting of a hinge region, a transmembrane (TM) and an intracellular (IC) site. The chains were fused. Connecting Peptide-MHC to the entire TCR signaling machinery provides a more physiological signal. This MHC-TCR chimera is referred to as MCR in the context of this specification. Such molecules, upon transfection into TCR-deficient T cell hybridomas, allow direct observation of peptide-MHC binding by TCRs of specific T cells using the NFAT-EGFP reporter system (Fig. 2a). By co-culturing cells with a peptide-MHC molecule library with antigen-specific T cell clones or hybridomas, a large-scale, parallel functional screen in mammalian cells can be used to directly determine the homologous peptide specificity of T cells. Can be identified.

Therefore, MCRs are designed and cloned for the screening of allogeneic peptides on MHC class II restricted T cells. Hereinafter referred to as MCR2. MCR2 the two chains: alpha chain containing extracellular part of the ^I-A b MHC class IIα chain linked to the truncated TCR a, as well as extracellular site of ^I-A b MHC class IIβ chain joined to truncated TCRβ and It consists of two chains, the β chain, which contains the peptide (epitope from major LCMV, gp61) (FIG. 1a). A second MCR2 was also cloned carrying an OVA peptide, the two were designated MCR2 (gp61) and MCR2 (OVA), respectively. After transduction of MCRs into MHC-II ^- TCR ^- BEKO thymoma cell line, their expression was confirmed by staining with anti-MHC-II antibody. As shown in FIG. 1b, the MCR2 was efficiently expressed on the cell surface. This indicates association with the CD3 component of the TCR complex. To confirm specificity, BEKO cells expressing MCR2(gp61) or MCR2(OVA) were co-cultured with purified Smarta2 or OT-II CD4 ⁺ T cells. Very early, a surface-specific down-regulation of peptide-specific MCR2 was observed with the same kinetics as conventional TCRs. MCR2 (gp61) was down-regulated when co-cultured with Smarta2 T cells, but not in the presence of OT-IIT cells (Fig. 1c left panel). The reverse was also true for MCR2 (OVA), highlighting the specificity of the MCR2 system. We further assessed the ability of MCR2 to elicit NFAT activation by transducing TCR-deficient T cell hybridomas carrying the NFAT-EGFP reporter (H18.3.13). Again, NFAT responses were elicited only when MCR2-bearing hybridomas were co-cultured with peptide-specific T cells. The response was already strong and easily measurable after 2 hours (Fig. 1d rightmost panel).

We mixed MCR2(gp61) ⁺ and MCR2(OVA) ⁺ reporter cells at different ratios and measured MNF by measuring NFAT activation after co-culture with Smarta2 or OT-II CD4 ⁺ T cells. The sensitivity of the system was tested. As shown in FIG. 1e, it was possible to directly detect the specific NFAT reporter expression in cells present at frequencies above 1/10000. Importantly, a linear correlation was observed between the percentage of detected NFAT-EGFP expressing cells and the percentage of "T cell/idotype-specific" MCR2 bearing cells, even if the Specific cells present at frequencies below 1/10000, even though indistinguishable from ground, were still shown to be NFAT-EGFP ⁺ . The lowest frequency of peptide-specific T cells in the heterogeneous population that was able to elicit robust NFAT activation in MCR2 ⁺ cells was also determined. CD4 ⁺ T cells isolated from Smarta2 and OT-II mice (FIG. 1f upper panel) or unsorted spleen cells (FIG. 1f lower panel) were mixed in different ratios to give MCR2 (gp61) ⁺ or MCR2 (OVA). ) ⁺ Used to stimulate cells. Even as little as 1% of peptide-specific CD4 ⁺ T cells evoked 50% of the maximal value of NFAT-activation in MCR2 ⁺ cells when T cells were provided in excess. Remarkably, as shown in FIG. 1g (left panel), even single Smarta2 CD4 ⁺ T cells were able to elicit significant NFAT-EGFP activation in MCR2(gp61) ⁺ cells, whereas In the case of MCR2(OVA) ⁺ cells, 5 cells are required (FIG. 1, right panel), probably because of the low affinity of the interaction. These results indicate that MCR technology can be used as a sensitive diagnostic tool for observing the specificity of T cells in blood drawn from patients. Indeed, MCR2(gp61) ⁺ reporter cells can be used to efficiently follow the proliferation of antigen-specific CD4 ⁺ T cells in the blood of LCMV infected animals (FIG. 1h). These results show the excellent sensitivity of the MCR method.

In order to use the disclosed invention to find rare specific peptides in complex libraries, multiple cycles of NFAT-EGFP ⁺ reporter cell co-culture and isolation are required. Efficient detection of NFAT activation in subsequent rounds of stimulation relies on the rapid disappearance of the NFAT reporter signal evoked in previous rounds, so that the very stable EGFP replaces the slow fluorescence timer (sFT). did. This mCherry mutant changes its "color" over time and can distinguish between new (blue-mCherry) and old (red-mCherry) NFAT activation over time, and thus more closely spaced subsequent stimulations. Can be shortened.

First, the disclosed invention was applied to search for gp61 mimotopes in MCR2 (gp61-RSS) libraries generated by randomization of the central residues of gp61 through RAG-mediated reconstitution (Fig. 2a and material. And method). Randomly selected clones consistently contained a unique mutant of the gp61 sequence (Fig. 2a) after transduction of this library into NFAT-sFT-bearing reporter cells (16.2c11). MCR2 ⁺ cells were isolated, expanded and co-cultured with gp61-specific or OVA-specific T cell hybridomas (FIG. 2b). About 10% of MCR2(gp61-RSS/IA ^b ) ⁺ 16.2c11 cells showed blue NFAT-reporter activation when co-cultured with gp61 specific hybridoma. These cells were isolated as single cells, expanded and rescreened (Figure 2c). All 24 clones tested responded to restimulation with gp61-specific hybridomas. PCR amplification and sequencing of the peptide portion of MCRs from those clones revealed two new mimotopes and the original gp61 peptide (Fig. 2c). Lysine 9 of Gp61 was mutated to serine (gp61S) or asparagine (gp61N), indicating that it is not required for Smarta2 T cell activation. In co-culture of dendritic cells and T cells, both mimotopes induced a robust Smarta2 T cell response. Interestingly, gp61N induces proliferation and production of Th1-like cytokines, similar to the original peptide, while suboptimal gp61S was less effective in promoting proliferation but had a strong Th2-like cytokine response. Was induced.

Finally, CD4 ⁺ T cell hybridomas taken from LCMV-infected animals 5 and 8 days post infection were used to screen for novel LCMV epitopes (FIGS. 3a and b). The hybridomas carry the NFAT-EGFP reporter, which allows the demonstration of reactivity to LCMV and selects gp61 non-reactive hybridomas for further analysis (FIG. 3b). Five such hybridomas (strongly responsive H30, H14, H2 and weakly responsive H4, H3) live on the MCR2 molecule containing all possible overlapping peptides of LCMV glycoprotein (GP) and nuclear protein (NP). The rally was used to screen transduced 16.2c11 reporter cells. This "pure peptide library" (GPL) was created by cloning random fragments of cDNA encoding GP and NP into the MCR2 vector (Fig. 3c), and most of the recovered peptides were (1/6) natural. Representing proteins has significant advantages over random or combinatorial peptide libraries. In fact, for 4 out of 5 hybridomas, the LCMV-specific target peptide was directly identified. Three of the hybridomas (H30, H14 and H2) recognized the known major LCMV epitope NP311 and H3 reacted with the new epitope NP547 (Fig. 3d). The fifth hybridoma (H4) failed to obtain enrichment for reactive reporter cells even after three rounds of screening. The hybridoma was tested for TCR surface expression and found to be TCR negative. The TCR was probably lost during amplification after the initial LCMV-specific screen. This result further demonstrates the TCR specificity of the disclosed method. One additional hybridoma (H9) was used to screen an MCR2 library carrying random peptides and found several strongly reactive peptides, which were not similar to any LCMV peptide. .. This further supports that the strategy using GPL is better than the random peptide library for T cell epitope screening.

Disclosed herein is a novel molecular sensor, which enables detection of peptide-MHC-TCR interactions on the APC side with excellent specificity, sensitivity and fast rate. Using this reporter, a new approach has been established for unbiased and functional T cell epitope screening. This combines the versatility of expression cloning with excellent sensitivity and the high-throughput performance of isolation of fluorescence activated cells, allowing for efficient iterative screening of peptide libraries in mammalian cells. All this offers significant advantages over the methods known in the art. First, thanks to the multivalent interaction between MCRs and TCRs, high and low affinity binding produces similar NFAT reporter signals (Fig. 1d), so low affinity ligands are hard to overlook. Indeed, despite the extensive study of LCMV epitopes, a novel epitope-NP547 could be identified. Second, the peptides are screened in the context of native TCR and MHC molecules expressed on the surface of human cells, so recombinant engineering of individual TCRs or mutagenesis of MHC is not required. Third, the MCR technology facilitates efficient screening of libraries highly enriched for peptides derived from pathogens/cells/tissues targeted by T cells of interest, as exemplified by LCMV viral peptide screening. To

The MCR-based approach provides a versatile, easy-to-use, and powerful method of identifying T cell antigen-specificity. As such, it may impact various areas of basic and clinical research. Defining the specificity of the defined effector tumor infiltrating T cells allows the discovery of new tumor antigens. Defining the specificity of autoreactive tissue-infiltrating T cells will aid in the development of antigen-specific therapies for autoimmune diseases. In this regard, MCR may also allow for efficient redirection of T cell effector function to peptide-specific T cells, possibly removing repertoires from unwanted specificity. Furthermore, screening of mimotope libraries will lead to the discovery of high affinity peptide variants and the development of sensitive flow cytometry-based tests for antigen reactivity of circulating T cells in the blood of patients.

Materials and methods
Mice C57/B16 mice were purchased from Charles River. Smarta2 and OT-II mice were bred in the RTH mouse facility.

The cell line Beko is a spontaneous thymoma cell line derived from a TCRβ-deficient mouse. The H18.3.13 reporter cell line contains the NFAT-EGFP reporter (4 of the minimal human IL-2 promoter, each containing three NFAT binding sites ACGCCTTCTGTATGAAACAGTTTTTCCTCC (SEQ ID NO:001) inserted upstream of the EGFP coding sequence). Carrying a copy) was made by retroviral transduction into TCR-B6T cells. The 16.2c11 reporter cell line was generated by transfecting a 16.2T cell hybridoma with a vector encoding the NFAT-sFT reporter construct and the mouse ecotropic retrovirus virus Slc7a1.

Production of hybridomas Isolated T cells or thymocytes were activated with plastic-conjugated anti-CD3ε and anti-CD28 antibodies in the presence of mouse IL-2 for 2-3 days. Equal amounts of activated T cells and TCR α-β-BW5147 fusion partners were fused with PEG-1500 and plated onto plates with limiting dilution in the presence of 100 mM hypoxanthine, 400 nM aminopterin and 16 mM thymidine (HAT). ..

Cloning of MCR2 The MCR2 α and β chains have been cloned by standard techniques and contain the following parts:
MCR2 α-chain: the MHC-II I-A ^b α-chain residues 1-208 linked to residues 87-137 of the TCR α-chain constant region by a GGSGGSAQ (SEQ ID NO:002) linker.
MCR2 β chain: AHCSGGSGGSAQ (SEQ ID NO: 003) the MHC-II IA ^b β chain 1-217 residues linked to the TCR β chain constant region (C1) residues 123-173 by a linker. In the MCR2 (gp61) residue, the DSs at positions 29 and 30 of the MHC-II portion were replaced by the amino acid sequence SG LNGPDIYKGVYQFKSV GSGGGSGGSDS (SEQ ID NO:004). In MCR2(OVA), the same residues were replaced by the amino acid sequence S ISQAVHAAHAEINEAGR GSGGSGGSGDS (SEQ ID NO:005, including the OVA peptide).

The reporter cell line and isolated thymocytes retrovirally transduced MCRα and MCRβ were cloned into the pMYiresGFP retroviral vector so that MCRβ replaced with GFP. Throughout the study, we used this vector (pMY-MCRαiresMCRβ) to generate MCRs containing various peptides and called them MCRs (“peptides”/“MHC haplotypes”). The retrovirus-containing supernatant was produced in the ecotropic Phoenix packaging cell line, infected with the reporter cell line and used to isolate cells.

To make the fabrication Gp61 mimotope library through RAG of mimotope library, MCR2 (gp61-RSS-EGFP -RSS / I-A b) construct, MCR2 (gp61) intermediate Gp61 peptide constructs in Was prepared by inserting a stuffer fragment containing the EGFP and RAG recombination signal sequences (RSS) into E. The structure was cultured on Tst-4 / DLL1 ^32, isolated ^CD4 - CD8 ^- transduced into double-negative (DN) thymocytes. After culturing for 1 week (during which, DN cells become CD4 ⁺ CD8 ⁺ double positive (DP) cells and their TCR genes recombine in the same manner as RSS-EGFP-RSS stuffer by RAG-mediated rearrangement). Cells expressing low levels were isolated and cDNA was made. The peptide coding portion of the recombinant MCR2 (gp61-RSS-EGFP- RSS / I-A b) and the cDNA or PCR amplification, MCR2 (gp61-RSS / I -A b) mimotope empty MCR2 generating libraries It was cloned into the ( ^IAb ) vector.

Generation and Screening of Pure and Random Peptide Libraries To generate MCR2-LCMV pure overlapping peptide libraries, DNA encoding GP and NP proteins was digested for a limited time (Takara DNA Fragmentation Kit). The fragment was ligated to the vector sequence flanking the cloning position with a homologous linker, PCR amplified, cloned into the pMY-MCR2 vector by Gibson assembly and transfected into bacteria producing 2×10 ⁶ clones. 16.2c11 cells were transduced with the library and 0.5×10 ⁶ MCR ^low cells and 2.2×10 ⁴ MCR ^high cells were isolated.

The MCR2 random peptide library was prepared by cloning an oligonucleotide (GGTNNNNNNNNTWCNNNNNNBCCCNNNNSCCNNNNNGGA) (SEQ ID NO:006) into the MCR2 vector using the above strategy. This oligonucleotide encoded a random amino acid at the position facing the TCR, but the anchor residues were partially fixed, ensuring good presentation. The complexity is 5.5×10 ⁶ bacterial clones and 11.5×10 ⁶ individual MCR2 ⁺ cells were isolated after transduction.

MCR Downregulation Assay Unless otherwise stated, MCR2 ⁺ Beko cells were co-cultured with a 5-fold excess of CD4 ⁺ T cells isolated from the indicated donor mice.

Stimulation of MCR ⁺ H18.3.13 or 16.2c11 cells Unless otherwise stated, MCR ⁺ cells were 5-fold excess of CD4 ⁺ T cells or CD4 isolated from the indicated donor mice. ⁺ T cell hybridomas were co-cultured for 8-12 hours.

Example 2: Chimeric antigen receptor polypeptide heterodimer first polypeptide, α chain (SEQ ID NO:007):
MPRSRALILGVLALTTMLSLCGGEDDIEADHVGTYGISVYQSPGDIGQYTFEFDGDELFYVDLDKKETVWMLPEFGQLASFDPQGGLQNIAVVKHNLGVLTKRSNSTPATNEAPQATVFPKSPVLLGQPNTLICFVDNIFPPVINITWLRNSKSVADGVYETSFFVNRDYSFHKLSYLTFIPSDDDIYDCKVEHWGLEEPVLKHWEPEGGSGGSAQSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
Amino acids 1-208 are derived from the MHC2α chain. Amino acids 209-216 are linker sequences. Amino acids 217-267 are from TCRα.

Second polypeptide (SEQ ID NO:008):
MALQIPSLLLSAAVVVLMVLSSPRTEGGSGGSGGSGDSERHFVYQFMGECYFTNGTQRIRYVTRYIYNREEYVRYDSDVGEHRAVTELGRPDAEYWNSQPEILERTRAELDTVCRHNYEGPETHTSLRRLEQPNVVISLSRTEALNHHNTLVCSVTDFYPAKIKVRWFRNGQEETVGVSSTQLIRNGDWTFQVLVMLEMTPRRGEVYTCHVEHPSLKSPITVEWRAQSGGSGGSAQGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS
Amino acids 1-26 are leader peptides. Between amino acids 27 and 28 is the insertion site for the presented oligopeptide. Amino acids 29-36 are linker sequences. Amino acids 37-228 are from MHC2β. Amino acids 229-236 are linker sequences. Amino acids 237-287 are from TCRβ.

Peptide inserted between amino acids 27 and 28 of SEQ ID NO:008 (GG):

Gp61 (SEQ ID NO: 009):
LNGPDIYKGVYQFKSV

OVA (SEQ ID NO: 010):
ISQAVHAAHAEINEEAGR

Claims (16)

A method for identifying a peptide sequence recognizable by TCR, comprising:
i. Providing a plurality of mammalian cells, wherein - at least two mammalian cells express members of the library;
-At least two members of said library encode transgenic antigen receptors;
-The transgenic antigen receptor is:
The extracellular position of MHC1 or MHC2,
An oligopeptide contained within said extracellular position, wherein said oligopeptide differs for at least two members of said library, and wherein said oligopeptide is presented in a manner suitable for recognition by the T cell receptor ,
· T cell transmembrane region of the receptor (TCR), and · TCR alpha chain of intracellular sites and TCRβ chains of intracellular sites,
Including;
Said transgenic antigen receptor is operably linked to a reporter protein, whereby binding of an allogeneic T cell receptor to said transgenic antigen receptor results in activation of said reporter gene,
ii. Contacting the plurality of mammalian cells with a preparation of T lymphocytes,
iii. Separating cells exhibiting a detectable reporter protein from the plurality of mammalian cells according to a detectable level of the reporter protein to obtain activated cells,
iv. Isolating DNA from the activated cells, and v. Determining the sequence of the oligopeptide sequence contained in the transgenic antigen receptor,
A method for identifying a peptide sequence recognizable by TCR, comprising: A method for identifying a peptide sequence recognizable by TCR, comprising:
i. Providing a mammalian cell, wherein said mammalian cell expresses a transgenic antigen receptor;
The transgenic antigen receptor molecule comprises: an extracellular position of MHC1 or MHC2 comprising an oligopeptide presented in a manner suitable for recognition by a T cell receptor,
· T cell transmembrane region of the receptor (TCR), and · TCR alpha chain of intracellular sites and TCRβ chains of intracellular sites,
Including;
Said transgenic receptor is operably linked to a reporter protein, whereby binding of an allogeneic T cell receptor to said transgenic antigen receptor results in activation of said reporter gene,
ii. Contacting said mammalian cells with a preparation of T lymphocytes, thereby activating said reporter gene to obtain activated mammalian cells,
iii. Isolating DNA from the activated mammalian cells, and iv. Determining the sequence of the oligopeptide sequence encoded in the transgenic chimeric antigen receptor,
A method for identifying a TCR recognition peptide sequence, comprising: Said transgenic antigen receptor is a chimeric antigen receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, wherein a. The first polypeptide comprises the extracellular portion of the major histocompatibility complex I (MHC class I) α chain, and the second polypeptide comprises a β2 microglobulin site, or
b. The first polypeptide comprises the extracellular portion of the major histocompatibility complex II (MHC class II) α chain, and the second polypeptide is the major histocompatibility complex II (MHC class II) β chain. Including the extracellular portion of
Here, at least one of the first and second polypeptide chains further comprises an oligopeptide covalently linked to the extracellular MHC site; 1 of the first polypeptide and the second polypeptide One further comprises an intracellular site or intracellular tail of the T cell receptor α chain, a transmembrane site, and a hinge region, and the other of the first polypeptide and the second polypeptide is a T cell Including intracellular portion of receptor β chain, transmembrane portion and hinge region,
The method according to claim 1 or 2. A chimeric antigen receptor polypeptide heterodimer comprising a first polypeptide and a second polypeptide, wherein:
a. The first polypeptide comprises the extracellular portion of the major histocompatibility complex I (MHC class I) α chain, and the second polypeptide comprises a β2 microglobulin site, or
b. The first polypeptide comprises the extracellular portion of the major histocompatibility complex II (MHC class II) α chain, and the second polypeptide is the major histocompatibility complex II (MHC class II) β. Including the extracellular portion of the chain,
One of the first polypeptide and the second polypeptide further comprises an intracellular site or intracellular tail of the T cell receptor α chain, a transmembrane site and a hinge region, and the first polypeptide The other of the peptide and the second polypeptide comprises an intracellular site, a transmembrane site and a hinge region of the T cell receptor β chain,
A chimeric antigen receptor polypeptide heterodimer comprising the first polypeptide and the second polypeptide. 5. At least one of said first and said second polypeptide chains further comprises an oligopeptide recognizable by a T cell receptor, said oligopeptide being covalently linked to said extracellular MHC site. A chimeric antigen receptor polypeptide heterodimer comprising the first polypeptide and the second polypeptide according to 1. The extracellular part of the MHC molecule
i. Each of MHC class I α1, α2 and α3 forming the first polypeptide portion, or
ii. MHC class IIα1 and MHC class IIα2 forming the first polypeptide part, and MHC class IIβ1 and MHC class IIβ2 sites forming the second polypeptide part, and/or
iii. Where the extracellular portion of the MHC molecule is selected from members of the human major histocompatibility complex gene family HLA as described in the IMGT HLA database,
The chimeric antigen receptor polypeptide heterodimer according to claim 4 or 5, which comprises: The oligopeptide sequence is
a. Inserted 1, 2, 3, 4 or 5 amino acids after the MHCα1 site sequence, and/or b. The oligopeptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 amino acids in length,
A chimeric antigen receptor polypeptide heterodimer according to any one of claims 4-6. Second having an amino acid sequence having a least be 90% of the first polypeptide and least be 90% identity to SEQ ID NO: 008 has an amino acid sequence having identity to SEQ ID NO: 007 The chimeric antigen receptor polypeptide heterodimer according to any one of claims 4 to 7, which comprises the polypeptide of. A nucleic acid molecule encoding the chimeric antigen receptor polypeptide heterodimer according to any one of claims 4-8. A cell,
i. 9. The chimeric antigen receptor polypeptide heterodimer of any one of claims 4-8, and ii. Said cell comprising an effector function operably linked to said chimeric antigen receptor polypeptide heterodimer. The effector functions are:
i. A reporter protein, and/or ii. A protein capable of inducing cell death in a cell bound to the chimeric antigen receptor polypeptide,
The cell according to claim 10, which is: The reporter protein is:
iii. Fluorescent protein, or iv. A luciferase protein, or v. A protein encoded by an antibiotic resistance gene, or vi. Cre recombinase, or vii. CAS-9 nuclease, or viii. Selected from a CAS-9 chimeric transcription repressor or transcription activator,
The cell according to claim 11. A method for obtaining a preparation of T lymphocytes having reduced reactivity to an antigen, comprising:
i. Providing a preparation of T lymphocytes obtained from a patient,
ii. Contacting the preparation of T lymphocytes with a preparation of cells according to any one of claims 10 to 12, comprising a chimeric antigen receptor polypeptide heterodimer.
Here, the cell is a cell bound to a chimeric antigen receptor polypeptide heterodimer, wherein the effector function induces cell death,
A method for obtaining a preparation of T lymphocytes having reduced reactivity with an antigen, which comprises the above steps. 14. Any one of claims 10 to 13 for use in the treatment or prevention of autoimmune disease or immune dysfunction, including transplant rejection, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, Crohn's disease and inflammatory bowel syndrome. The cell according to the item. A method for detecting an immune response to a patient's antigenic peptide, comprising:
i. Providing a cell according to any one of claims 10 to 12, wherein the cell comprises a reporter protein operably linked to the chimeric antigen receptor polypeptide heterodimer.
ii. Providing the patient's blood sample ex vivo,
iii. Contacting the mammalian cell with the blood sample, and iv. Detecting the reporter protein in the mammalian cell,
Including the above steps,
A method for detecting an immune response to a patient's antigenic peptide. i. Separating the mammalian cells according to expression of a reporter protein,
ii. Isolating DNA from the separated mammalian cells, and iii. Determining the sequence of the antigen-peptide encoded in the chimeric antigen receptor polypeptide heterodimer according to any one of claims 4-8.
Further comprising the above steps,
The method according to claim 15.